Lignin is an aromatic polymer that is deposited in secondary-thickened cells where it provides strength and impermeability to the wall. In dicot plants, lignin is mainly composed of the monolignols coniferyl and sinapyl alcohol that give rise to the guaiacyl (G) and syringyl (S)-units of the lignin polymer, respectively. In addition, a number of other units may be incorporated at lower levels, depending on the species, the genetic background and environmental conditions (Ralph et al., 2004). The lignin biosynthetic pathway is generally divided in two parts: the general phenylpropanoid pathway from phenylalanine to feruloyl-CoA, and the monolignol-specific pathway from feruloyl-CoA to the monolignols (FIG. 1). Ten enzymes are involved in the pathway from phenylalanine to the monolignols:phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeoyl-CoA O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD) (FIG. 1) (Boerjan et al., 2003; Bonawitz and Chapple, 2010). After their biosynthesis, the monolignols are translocated to the cell wall where they are oxidized to radicals, which are further coupled in a combinatorial fashion with the formation of various types of chemical bonds where the ether (8-O-4), resinol (8-8) and coumaran (8-5) bonds are the most prominent ones (Ralph et al., 2004). The possibility of obtaining plants with an altered amount or structure of lignin by mutant screening (Vermerris et al., 2007) or genetic engineering (Chen and Dixon, 2007) has enabled the properties of plant biomass to be improved for forage digestibility, or processing into pulp or fermentable sugars (Pilate et al., 2002; Baucher et al., 2003; Chen and Dixon, 2007). A remarkable insight from this work is that plants with reduced lignin can either grow normally, or have dramatic effects on growth and development, depending on which gene of the lignin biosynthetic pathway that was perturbed. Apparently, plants are sometimes able to successfully cope with a mutation, a phenomenon called phenotypic buffering, while in other cases, they are not (Fu et al., 2009).
In an attempt to understand how plants cope with a genetic defect, it was previously shown that altering the expression of genes in the lignin biosynthetic pathway not only results in altered lignification, but also in shifts in both primary and secondary metabolism. For example, reductions in lignin in Arabidopsis pal1 pal2 double mutants, while not leading to abnormalities in overall plant growth, were accompanied by transcript changes of genes involved in phenylpropanoid biosynthesis, carbohydrate metabolism, stress-related pathways, signal transduction and amino acid metabolism, as studied by cDNA-AFLP (Rohde et al., 2004). Whereas all identified phenolic compounds were lower in abundance, nearly all of the detected amino acids accumulated in these mutants. Analogous experiments with CCR-down-regulated poplar showed an induced stress response and effects on cell-wall biosynthetic genes, including the induction of transcripts for PAL and reduction of hemicellulose and pectin biosynthesis. Metabolite analysis not only showed major increases in phenolic acid glucosides, but also shifts in the primary metabolites, e.g., increased levels of maleate, Kreb's cycle intermediates and several monosaccharides (Leple et al., 2007). Similar effects were observed in CCR-down-regulated tobacco where several amino acids accumulated (Dauwe et al., 2007). In CAD-down-regulated tobacco, most transcripts of genes involved in phenylpropanoid biosynthesis were lower and transcripts of light- and (non-lignin) cell wall-related genes were higher (Dauwe et al., 2007). Microarray analysis of cad-c cad-d Arabidopsis mutants revealed effects on stress-related pathways and cell wall-related proteins, including lignin, pectin, cellulose and cell wall-localized proteins (Sibout et al., 2005). Furthermore, an SSH transcript-based comparison of three brown midrib 3 (bm3) maize mutants (mutated in COMT), revealed a feedback on phenylpropanoid and hemicellulose biosynthesis and photosynthesis, and comparisons with bm1 (mutated in CAD) and bm2 (mutated gene unknown) showed a shared response in signaling and regulation (Shi et al., 2006a). So far, these types of studies have remained fragmentary and disconnected, focusing on a few individual genes in different species and the transcript and metabolite profiling methods used did not allow the extraction of genome and metabolome-wide conclusions. Hence, the major adjusting and regulatory mechanisms that may exist across the pathway to compensate for either less or modified lignin have remained largely unresolved (Vanholme et al., 2008; Vanholme et al., 2010c; Vanholme et al., 2010a). For example, it is unclear whether the flux through the phenylpropanoid pathway is redirected in a systematic way upon blocking particular steps in monolignol synthesis and to what extent feedback systems regulate the pathway. To obtain deeper insight into lignin biosynthesis and the metabolic network it is embedded in, a systems biology approach was used as defined by Ideker et al. (2001), i.e., the study of the consequences of pathway perturbations, followed by computational analysis of the data. To this end, the transcriptome and metabolome of mutants in consecutive steps of the lignin biosynthetic pathway were systematically analyzed. The spectacular advances in transcriptomics and metabolomics have opened up the possibility of fully exploiting this approach (Oksman-Caldentey and Saito, 2005; Mochida and Shinozaki, 2011), as already illustrated by studies of the consequences of altered expression of five transcription factors involved in glucosinolate biosynthesis in Arabidopsis (Hirai et al., 2007; Malitsky et al., 2008). Arabidopsis was chosen as a model because systems biology can only be properly performed in an organism for which thorough basic knowledge exists on the identity and function of genes, proteins and metabolites. Furthermore, the inflorescence stem was focused on, which is an excellent model for wood formation (Nieminen et al., 2004) as it is rich in fibers and vessels, both cell types undergoing lignification during secondary thickening. The set of Arabidopsis lines studied were mutated in PAL1 and 2, C4H, 4CL1 and 2, CCoAOMT1, CCR1, F5H1, COMT and CAD6, genes predicted to be involved in developmental lignification (Boerjan et al., 2003; Costa et al., 2003; Goujon et al., 2003a; Raes et al., 2003; Bonawitz and Chapple, 2010). It was shown that most of the mutations provoked a strong and organized response at the transcript and metabolite levels, even when these mutants did not have any apparent visible phenotype. Mutants with reduced lignin levels up-regulated genes of the pathways that supplied monolignol precursors, whereas mutants with compositional shifts down-regulated these pathways. In addition, metabolic profiling showed that perturbations redirected the flux into novel pathways, at the same time revealing metabolic rerouting of accumulating metabolites by 4-O and 9-O hexosylation.